Activated platelets can induce myocardial infarction and cerebrovascular thrombosis, whereas insufficient platelet activation can result in pathological blood loss. A better understanding of the complex network of events that govern platelet activation could lead to the identification of new therapeutic targets that mitigate thrombosis without significantly increasing bleeding. We previously used a novel chemoproteomic approach to identify arylacetamide deacetylase-like 1 (AADACL1) as a lipid hydrolase in human platelets. We found that AADACL1 is required for agonist-stimulated platelet aggregation and thrombus formation but not platelet adhesion ex vivo. Exciting new data indicate that AADACL1 inhibition significantly prolongs occlusion time in a rat model of thrombosis, suggesting that blocking AADACL1 activity in vivo may effectively reduce thrombosis while preserving other aspects of hemostasis, a hypothesis that we will test in Specific Aim 1. Even though AADACL1 activity contributes to the signaling output of multiple platelet agonists, we have only a cursory understanding of its substrates, products and mechanism of action in platelet aggregation. Obtaining this fundamental knowledge is crucial for evaluating the scope of AADACL1 action in platelets and determining the prospective value of AADACL1 and its substrates, and products as potential regulators of thrombosis. Therefore, in Specific Aim 2 we propose to define the metabolic footprint of AADACL1 in platelets via global and targeted mass spectrometry-based lipidomics. Finally, we determined that AADACL1 is required for optimal activation of Rap1 and PKC, two platelet signaling nodes critical to the platelet activation process in response to multiple agonists. Although the mechanism by which AADACL1 regulates these key signaling nodes is unknown, our intriguing preliminary data suggest that ether lipids metabolized by AADACL1, may function as endogenous regulators of platelet signaling at least in part via direct interaction with the C1 domains of PKC, a hypothesis that will be tested in Specific Aim 3. Completion of these studies will help us dissect an entirely novel lipid signaling pathway in platelets and probe the biological implications of AADACL1 metabolism in thrombosis and hemostasis.